Cancer affects tens of millions of people worldwide and is a leading threat to public health in the United States. As the new generation of cancer therapy, immunotherapy has undergone significant progress in this decade, including the successful utilization of adoptive T cell therapy and immune- modulatory drugs to treat melanoma, kidney cancer, lung cancer and leukemia. These breakthroughs mark a new era for cancer immunotherapy and set the stage for its fast expansion. Among the candidate immunotherapy agents, invariant natural killer T (iNKT) cells are especially attractive due to thei immediate and potent responses to stimulations and their capacity to target various types of cancer independent of tumor antigen restriction. However, the application of iNKT cell-based therapies has been greatly hindered by the low frequency and high variability of iNKT cells in humans (~0.001-1% in blood). Innovative approaches that can overcome these limitations are therefore desperately needed. This project aims to develop a novel iNKT cell therapy for cancer that overcomes the current limitations by genetically engineering hematopoietic stem cells (HSCs) to produce iNKT cells targeting cancer. Because of the longevity and self-renewal of HSCs, this new therapy has the potential to provide patients with therapeutic levels of engineered iNKT cells for a lifetime. In order to demonstrate this novel therapy, a proof-of-principle study using a conventional mouse model will be performed to analyze its feasibility and efficiency in targeting syngeneic mouse melanoma. To facilitate the translation of the therapy to humans, a specialized mouse model harboring the human immune system will be employed to investigate the therapeutic potential of human HSC-engineered iNKT cells against human melanoma. At the heart of this project is the concept known as SEI (stem cell- engineered immunotherapy). As the inventor of SEI and multiple other innovative immunotherapy approaches, the PI is uniquely suited to accomplish this project. The PI's demonstrated track record of productive translational research will also ensure the successful transition of this nove therapy from bench to bedside. Once established, this new therapy can be readily adapted to treat cancers other than melanoma. Moreover, although this project focuses on cancer, iNKT cells have been suggested to regulate many other human diseases, including infections such as tuberculosis, allergies such as asthma, and autoimmune diseases such as Type I diabetes and multiple sclerosis. The success of this project will thus enable the future application of stem cel-engineered iNKT cell therapy for treating many diseases and have a substantial impact on public health.